Along with the large scale planting of the transgenic plants, increasing attentions have been paid to the biological confinement of transgenic plants. It was observed that a gene encoding glyphosate-tolerant CP4 EPSPS could spread to crops 20 kilometers away from the transgenic plants, even into weeds, by Wruad et al. Proc Natl Acad Sci USA 2004, 101, (40), 14533-8. Thus it is very important to perform biological confinement towards transgenic plants for preventing superweeds and the like.
Modes of exogenous gene spreading of transgenic plants comprise principally the following: diffusion of the pollen from transgenic plants; transgenic plants acting as the recipients of pollen from wild sibling species to form hybrids; gene spreading which may be caused by the DNA of transgenic plants. Recently there are some methods which control the exogenous gene spreading of transgenic plants, e.g., (1) physical isolation, mainly distant isolation, so that the exogenous gene spreading by pollen is blocked; (2) genetic control, including: (a) male sterility; (b) genome incompatibility, that is, a specific exogenous gene is integrated into the crop genome which is incompatible with weeds; (c) maternal inheritance, which has achieved primary success in tobacco (Daniell et al., Nat Biotechnol 1998, 16, (4), 345-8), where exogenous genes are introduced into the chloroplasts of plants and subjected to maternal inheritance, thus no spreading into other species by diffusing pollen; (d) seed sterility; (e) transgenic mitigation (TM), where TM genes which are closely linked with target genes, advantageous or neutral to transgenic plants and disadvantageous to the growth of weeds (such as to prevent seeds from spilling out and to reduce the secondary dormancy of seeds) are used to prevent the production of superweeds.
A novel method for controlling the transgene flow of glyphosate-tolerant gene was reported by Ye, G. N. et al. Plant J 2001, 25, (3), 261-70 and Chin, H. G. et al. Proc Natl Acad Sci USA 2003, 100, (8), 4510-5. In this method, a EPSPS gene was divided into two fragments which were then linked respectively with a gene expressing DanE intein and were co-expressed. A full length EPSPS was formed by self splicing of intein. The resulting E. coli or tobacco becomes tolerant to glyphosate. However, the intein-encoding gene itself was introduced into transgenic plants in this method, which might also lead to additional risks such as transgene flow.
Protein fragments derived from proteolytic cleavage or gene expression reconstitute in vivo or in vitro into a complex with the same function as the intact protein. This is known as protein fragment complementation or protein reconstitution technology (e.g., Hakansson, M. et al. Curr Protein Pept Sci 2002, 3, (6), 629-42; Braun, M. et al. J Bacteriol 2003, 185, (18), 5508-18). It is indicated by a research on fragment complementation of aminoacyl tRNA synthetase that most divisional sites of protein fragment for fragment complementation locate in non-conserved regions. That a protein can be reconstituted means the non-covalent interaction within this protein is quite specific, which makes the protein segments advantageous in forming a natural structure (Shiba, K. et al. Proc Natl Acad Sci USA 1992, 89, (5), 1880-4; Shiba, K. et al. J Biol Chem 1992, 267, (32), 22703-6). Furthermore, functional reconstitution of a protein means that the protein keeps a relative stable structure, even if the covalent bond is broken. Various non-covalent interactions (such as hydrogen bond, salt bridge and hydrophobic interactions) exist between two peptides for fragment complementation and the interactions are important for retaining stable structure of the protein (Nelson, K. E. et al., Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ Microbiol 2002, 4, (12), 799-808).
We reconstituted active EPSPS in vivo and in vitro by such protein reconstitution. Glyphosate-tolerant EPSPS could also be reconstituted in this way. Fragment complementation of EPSPS can be used in transgenic plants to improve safety and reduce the possibility of superweed formation.